Characterization of Cathlelicidin gene in river buffalo
Egyptian river buffalo tissues such as lung, trachea, muscle, intestine, liver, mammary gland, ovary, colon, testis, tongue, kidney, lymph node and blood, were screened for the presence of Cathlelicidin (Cathl) gene transcripts. Based on published Bubalus bubalis Cathl cDNA, primer pairs were designed to amplify a mRNA segment of 500bp that includes the full coding region (444bp). Cathl was expressed in trachea, lung, mammary gland, ovary, lymph node and liver. Alignment of the 500bp nucleotide full spliced Cathl mRNA of buffalo trachea and lung revealed two SNPs at nt-52(C/T) and at nt-179(C/A), of the coding region (sequences were submitted to NCBI/ GenBank with accession numbers KC354786 and AB675411, respectively). The nt-52 SNP results in two codons CTG and TTG which were both translated to Leucine, a silent mutation. SNP at nt-179 results in the two codons GAC/GAA translated to glutamic acid in trachea versus aspartic acid in lung which is a conserved amino acids substitution. Variations in amino acids of Cathl cDNA between Egyptian buffalo and Indian buffalo, which are also of the river type, have been investigated. The extent of intron retention in Cathlelicidin has been investigated in Egyptian buffalo Cathl cDNA, since intron-I retention has been reported previously. A set of 3 primer pairs were designed from Bos taurus DNA to amplify segments that cover parts of every two successive exons. The first primer pair amplified a 248 bp segment in lung. However it amplified a 353 bp segment in other tissues including the trachea which revealed the presence of intron-I (105 bp). A second primer pair, designed to amplify a 275bp in cattle DNA, amplified a fragment of 275bp in cDNA of buffalo lung, mammary gland, trachea, ovary, tongue, colon, testis, intestine, liver and blood; revealing the retention of intron-II (131bp). However retention of intron III in Egyptian buffalo was not verified. The results indicate that Cathl occurs in Egyptian buffalo in two mRNA variants: a fully spliced and introns-retaining isoforms.